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Journal of Virology, March 2005, p. 3277-3288, Vol. 79, No. 6
0022-538X/05/$08.00+0     doi:10.1128/JVI.79.6.3277-3288.2005
Copyright © 2005, American Society for Microbiology. All Rights Reserved.

Deletion of Highly Conserved Arginine-Rich RNA Binding Motif in Cowpea Chlorotic Mottle Virus Capsid Protein Results in Virion Structural Alterations and RNA Packaging Constraints

Padmanaban Annamalai,1 Swapna Apte,1,{dagger} Stephan Wilkens,2 and A. L. N. Rao1*

Department of Plant Pathology,1 Department of Biochemistry, University of California, Riverside, California2

Received 5 August 2004/ Accepted 20 October 2004

The N-proximal region of cowpea chlorotic mottle virus (CCMV) capsid protein (CP) contains an arginine-rich RNA binding motif (ARM) that is also found in the CPs of other members of Bromoviridae and in other RNA binding proteins such as the Tat and Rev proteins of human immunodeficiency virus. To assess the critical role played by this motif during encapsidation, a variant of CCMV RNA3 (C3) precisely lacking the ARM region (C3/{Delta}919) of its CP gene was constructed. The biology and the competence of the matured CP derived in vivo from C3/{Delta}919 to assemble and package progeny RNA was examined in whole plants. Image analysis and computer-assisted three-dimensional reconstruction of wild-type and mutant virions revealed that the CP subunits bearing the engineered deletion assembled into polymorphic virions with altered surface topology. Northern blot analysis of virion RNA from mutant progeny demonstrated that the engineered mutation down-regulated packaging of all four viral RNAs; however, the packaging effect was more pronounced on genomic RNA1 and RNA2 than genomic RNA3 and its CP mRNA. In vitro assembly assays with mutant CP subunits and RNA transcripts demonstrated that the mutant CP is inherently not defective in packaging genomic RNA1 (53%) and RNA2 (54%), but their incorporation into virions was competitively inhibited by the presence of other viral RNAs. Northern blot analysis of RNA encapsidation in vivo of two distinct bromovirus RNA3 chimeras, constructed by exchanging CPs having the {Delta}919 deletion, demonstrated that the role of the conserved N-terminal ARM in recognizing and packaging specific RNA is distinct for each virus.

* Corresponding author. Mailing address: Department of Plant Pathology, University of California, 3264A Webber Hall, Riverside, CA 92521-0122. Phone: (951) 827-3810. Fax: (951) 827-4294. E-mail: a.rao{at}ucr.edu.

{dagger} Present address: Department of Biological Sciences, Purdue University, West Lafayette, IN 47907.

Journal of Virology, March 2005, p. 3277-3288, Vol. 79, No. 6
0022-538X/05/$08.00+0     doi:10.1128/JVI.79.6.3277-3288.2005
Copyright © 2005, American Society for Microbiology. All Rights Reserved.



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